NO* (nitric oxide) produced chemically or enzymatically within the host plays an essential role in innate immunity. An important property of NO* is its ability to inhibit the growth of a broad range of pathogenic microbes. This project examines NO* interactions with the essential transition metals iron (Fe) and zinc (Zn) and their contribution the antimicrobial actions of NO* in the enteric pathogen Salmonella Typhimurium. The central hypothesis is that host NO* restricts bacterial replication by disrupting metal homeostasis. The specific aims of the project are to: (1) Investigate the role of NO*-induced Fe efflux in Salmonella stress resistance - We have discovered that NO* induces Salmonella Fe efflux via a transporter called IceT (Iron-citrate efflux Transporter), which arrests growth and confers resistance to NO*, oxidative stress and antibiotics. Structural determinants of IceT function will be analyzed, and the relative contribution of IceT and other homologous (YieO) and non-homologous (FetAB) Fe efflux transporters to these phenotypes will be investigated. The roles of these transporters to Salmonella virulence and antibiotic killing in vivo will be determined. (2) Analyze the role of Zn transporters during nitrosative stress - We have found that NO* targets multiple Zn-containing metalloproteins and mobilizes free intracellular Zn. This aim will test the hypothesis that Zn stress plays an important role in NO*-mediated antimicrobial actions. The effects of NO* on Zn compartmentalization and the expression of genes involved in Zn uptake and efflux will be measured. Salmonella mutants lacking specific Zn-responsive regulators (Zur, ZntR), Zn uptake systems (ZnuABC, ZupT) or Zn efflux systems (ZntA, ZntB, ZitB, YiiP) will be used to determine whether Zn is an important determinant of NO*-mediated cytotoxicity in Salmonella, both in vitro and during infection in vivo. (3) Assess the ability of Z-binding proteins to prevent mismetallation during nitrosative stress - The expression of two novel homologous Zn-binding proteins (STM1808, YeaR) has been found to be strongly induced by NO*. The ability of these proteins to prevent mismetallation of Fe- S cluster-containing metalloproteins and to promote the recovery of Zn-containing metalloproteins following nitrosative stress will be assessed. These studies will provide novel mechanistic insights into NO*-mediated antimicrobial actions relating to transition metal homeostasis.